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Research Article
Integrative taxonomy reveals two new giant pill-millipedes of the genus Zephronia Gray, 1832 from eastern Thailand (Diplopoda, Sphaerotheriida, Zephroniidae)
expand article infoRuttapon Srisonchai, Natdanai Likhitrakarn§, Chirasak Sutcharit|, Thomas Wesener
‡ Khon Kaen University, Khon Kaen, Thailand
§ Maejo University, Chiang Mai, Thailand
| Chulalongkorn University, Bangkok, Thailand
¶ Zoological Research Museum Alexander Koenig, Leibniz Institute for the Study of Biodiversity Change (LIB), Bonn, Germany
Open Access

Abstract

A large amount of material of the millipede genus Zephronia Gray, 1832 was collected during 2014–2023 from many parts of eastern Thailand. An integrative study of morphological characters and genetic data (COI gene) revealed two new species: Z. chantaburiensis Srisonchai & Wesener, sp. nov. and Z. macula Srisonchai & Wesener, sp. nov. The two new species clearly differ from other congeners by their unique characteristics, especially in their colour pattern and telopod shape. The interspecific genetic distances of the 658 bp COI gene barcoding fragment between these new species and all other species of giant pill-millipede from Thailand, Laos and Cambodia are 12.01–23.49% for Z. chantaburiensis sp. nov. and 17.93–25.13% for Z. macula sp. nov. While relationships among species remain preliminary, the phylogenetic tree shows that species of Zephronia are interspersed with species of Sphaerobelum Verhoeff, 1924 and Prionobelum Verhoeff, 1924. Phylogenetic analyses place both new species in a clade termed Zephronia s.s., which receives support also from morphological data, showing a unique position of the organ of Tömösváry. Z. macula sp. nov. appears to occur over a broad distribution whereas Z. chantaburiensis sp. nov. was found only at the type locality. Given that all known records are in the eastern part of Thailand, we thus regard both species as endemic. Morphological illustrations based on SEM micrographs and a distribution map are also provided.

Key words

Biodiversity, limestone, Myriapoda, Southeast Asia, taxonomy

Introduction

Intensive research on the millipedes (class Diplopoda) in Thailand began in 2007 after the discovery of a beautifully ornamented and endemic creature, the shocking pink dragon millipede Desmoxytes purpurosea Enghoff, Sutcharit & Panha, 2007 which has been endorsed by the public and has inspired further studies on several millipede groups (Enghoff et al. 2007; Likhitrakarn et al. 2023). A large number of newly discovered species and unidentified specimens has become available since then, and many of these were recently described by conventional taxonomic methods and included in phylogenetic analyses (i.e., Likhitrakarn et al. 2011; Srisonchai et al. 2016; Pimvichai et al. 2020). Thailand presents the highest number of described diplopod species for SE Asia today, accounting for more than 264 of 500 known species in mainland Southeast Asia (Nguyen and Sierwald 2013; Likhitrakarn et al. 2023), with striking genera and species discovered in many underexplored locations of Thailand and even in anthropogenically modified habitats.

Currently, three genera of giant pill-millipedes (order Sphaerotheriida) are known from Thailand: Sphaerobelum Verhoeff, 1924 with four species (see Srisonchai et al. 2023), Prionobelum Verhoeff, 1924 with two species (see Donworth and Wesener 2024), and Zephronia Gray, 1832 with nine species (see Bhansali and Wesener 2022). Generally, Zephronia is one of the most speciose groups among the diplopods and contains more than 50 described species (Wesener 2016). Recent research has continued to underscore the remarkably high diversity within the genus Zephronia, which has led to the discovery of an increasing number of species (Golovatch et al. 2012; Semenyuk et al. 2018; Wesener 2019; Likhitrakarn et al. 2021; Srisonchai et al. 2021; Rosenmejer et al. 2021; Bhansali and Wesener 2022; Zhao et al. 2022). A total of nine species of Zephronia have been recorded so far from Thailand (see Likhitrakarn et al. 2023), while only one species is known from Cambodia (Z. dawydoffi Attems, 1953) and one from Laos (Z. laotica Wesener, 2019). However, as with other closely related genera within the family, taxonomic studies on the genus Zephronia have posed intriguing challenges due to the insufficient genetic information and unknown evolutionary relationships (Wesener 2019).

The genetic approach based on DNA barcoding has been widely used for species delimitation in millipedes in recent years, but it is considered more reliable when used in combination with morphological evidence. Previous phylogenetic studies have notably provided insights into species discrimination for Zephronia (Wesener 2019; Rosenmejer et al. 2021; Bhansali and Wesener 2022; Zhao et al. 2022).

Considering the newly collected material, two morphologically distinct groups of specimens were observed during fieldwork. Given their visible morphological differences from the other known Thai giant pill-millipede species, the suspicion arose that they may represent undescribed species. In this paper, we integratively describe two new species from the eastern part of Thailand based on morphological traits using scanning electron microscopy and phylogenetic analyses based on mitochondrial COI sequences, in order to confirm their status and compare them with the known congeneric species from Thailand, Laos and Cambodia.

Material and methods

Specimen collections

The material was obtained from the collections of the Chulalongkorn University Museum of Zoology (CUMZ). New sampling was also conducted throughout the eastern part of Thailand and the surrounding areas in 2022–2023. All individuals were collected by hand and some of them were photographed alive in the field using a Canon 90D digital camera equipped with a Canon EF 100 mm f/2.8 Macro USM lens. Animal euthanasia was applied during the management of specimens based on AVMA guidelines (American Veterinary Medical Association 2020). The research protocol was approved by the Institutional Animal Care and Use Committee, Khon Kaen University (No. IACUC-KKU-136/64). The studied specimens were preserved in 70% ethanol for morphological examination and in 95% ethanol for molecular analyses. The data of GPS and habitats were also recorded.

Maps

The map present herein was generated based on the background photo from the Elastic Terrain Map (Willett et al. 2015), and edited in Adobe Photoshop CS6.

Morphological identifications

The format of the morphological descriptions follows Golovatch et al. (2012), Wongthamwanich et al. (2012), Wesener (2019), and Semenyuk et al. (2018). More than 600 specimens were examined under the stereo microscope. All morphological characters were thoroughly compared with the previous descriptions and compared with some of the available type specimens or topotypes.

The holotypes, as well as the paratypes, are deposited in the Chulalongkorn University Museum of Zoology, Thailand (CUMZ), the Natural History Museum of Denmark, Denmark (NHMD), the Naturhistorisches Museum Wien, Austria (NHMW) and the Zoological Research Museum Koenig, Germany (ZFMK).

Abbreviations used in this study

List of abbreviations used in the description and figures:

3it 3-combed inner tooth of mandible,

bu bursa of vulva,

co condylus of mandible,

cp cuticular impression of endotergum;

Cp central pad of gnathochilarium,

cr-t crenulated teeth on telopods,

ct central tooth,

cx coxa,

et external tooth of epipharynx,

Et external tooth of mandibular gnathal lobe,

fe femur,

ia inner area of endotergum;

Ia inner area of mandible,

il incisura lateralis of the head,

imf immovable finger of telopod;

ip inner palpi of gnathochilarium,

ll lamellae lingulales of gnathochilarium,

ma middle area of endotergum;

me mentum of gnathochilarium,

ml membranous lobe,

mp molar plate of mandible,

op operculum of vulva,

pl pectinate lamellae of mandible,

pm posterior margin of endotergum;

po postfemur,

pre prefemur,

rsp row of spines,

sc sensory cone,

scl-s sclerotized spots,

st stipites of gnathochilarium,

st-pl stigmatic plate,

ta tarsus,

ti tibia.

Specimen repositories and others

CUMZ Chulalongkorn University Museum of Zoology, Thailand;

KKUMZ Khon Kaen University Museum of Zoology, Thailand;

NHMD Natural History Museum of Denmark, Denmark;

NHMW Naturhistorisches Museum Wien, Vienna, Austria;

ZFMK Zoological Research Museum Koenig, Bonn, Germany.

Scanning electron microscope

Body parts of specimens for scanning electron microscopy (SEM) were carefully dissected under a stereomicroscope, placed in a dry cabinet for 24 hours, mounted on aluminium stubs, and then coated with gold. Objects were examined under a high vacuum in JEOL, JSM-5410 LV at the faculty of Science, Khon Kaen University. All figures were assembled and adjusted in Adobe Photoshop CS6.

DNA extraction and PCR amplifications

DNA of 11 specimens (9 specimens of the two new species and 2 specimens of Z. siamensis) were extracted from the legs using the NucleoSpin Tissue Kit. We analysed the mitochondrial cytochrome c oxidase subunit I (COI) as a DNA barcoding gene. The polymerase chain reactions were carried out using LCO-1490 as forward and HCO-2198 as reverse primers — LCO-1490 (5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3') and HCO-2198 (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3') (Folmer et al. 1994). A total volume of 30.0 µL including 1.0 µL of DNA template — 43 °C for 2 minutes as annealing step and 72 °C for 2 minutes as an extension step. The target gene was verified by 1% agarose gel electrophoresis and eventually observed under UV trans-illumination. PCR products of COI were sequenced externally at Bioneers Co. (Korea).

DNA alignment and phylogenetic reconstruction

We analysed a total of 84 sequences comprising 11 sequences generated from this study and 73 sequences obtained from GenBank, including all known giant pill-millipede species of Zephronia from Thailand, Cambodia, and Laos. Sphaerobelum spp., Prionobelum spp., Cryxus ovalis (Linnaeus, 1758), Epicyliosoma sp., Arthrosphaera brandti (Humbert, 1865), Sphaeromimus splendidus Wesener & Sierwald, 2005 and Glomeris marginata (Villers, 1789) were used as outgroups. All new sequences were submitted to GenBank with accession numbers provided in Table 1.

Table 1.

Lists of Zephronia, Sphaerobelum, Prionobelum and Cryxus species analysed in this study and their COI accession numbers.

Species COI accession number Voucher code Locality Reference
Ingroups
1 Z. chantaburiensis sp. nov. PP754582 CUMZ (M47) Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim This study
2 Z. chantaburiensis sp. nov. PP754583 CUMZ (M48) Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim This study
3 Z. chrysomallos Bhansali & Wesener, 2022 OM509649 ZFMK MYR8826 Thailand, Kanchanaburi Province, Sai Yok District, Sai Yok Noi Waterfall Bhansali and Wesener 2022
4 Z. dawydoffi Attems, 1953 MK330971 ZFMK Myr4504 N/A Wesener 2019
5 Z. erawani Bhansali & Wesener, 2022 OM509650 NHMD K56x9 Thailand, Kanchanaburi Province, Si Sawat District, 50 km W of Kanchanaburi, Erawan Waterfall Bhansali and Wesener 2022
6 Z. golovatchi Srisonchai, Sutcharit & Likhitrakarn, 2021 OM509646 ZFMK MYR6262 Thailand, Nakhon Ratchasima Province, Pak Chong District Bhansali and Wesener 2022
7 Z. golovatchi Srisonchai, Sutcharit & Likhitrakarn, 2021 OM509647 ZFMK K53 Thailand, Nakhon Nayok Province, Khao Yai National Park Bhansali and Wesener 2022
8 Z. hui Liu & Wesener, 2022 OP339790 SCAU YGM03 China, Guizhou, Tongren City, Jiangkou County, Taiping Town, Yamugou Scenic Area Zhao et al. 2022
9 Z. hui Liu & Wesener, 2022 OP339791 SCAU YGM02 China, Guizhou, Tongren City, Jiangkou County, Taiping Town, Yamugou Scenic Area Zhao et al. 2022
10 Z. lannaensis Likhitrakarn & Golovatch, 2021 OM509629 ZFMK MYR3498 Thailand, Chiangmai Province, Mueang District, Doi Suthep Bhansali and Wesener 2022
11 Z. lannaensis Likhitrakarn & Golovatch, 2021 OM509630 ZFMK MYR3501 Thailand, Chiangmai Province, Mae Rim District, Traidhos School Campus Bhansali and Wesener 2022
12 Z. lannaensis Likhitrakarn & Golovatch, 2021 OM509631 ZFMK MYR4911 Thailand, Chiangmai Province, Mae Rim District, Mae Sa Valley Bhansali and Wesener 2022
13 Z. lannaensis Likhitrakarn & Golovatch, 2021 OM509632 NHMD K57B Thailand, Chiangmai Province, Mueang District, Doi Suthep, Me Sa Waterfall Bhansali and Wesener 2022
14 Z. lannaensis Likhitrakarn & Golovatch, 2021 OM509633 MHNG 3B Thailand, Chiangmai Province, Mueang District, Doi Suthep Bhansali and Wesener 2022
15 Z. laotica Wesener, 2019 MK330977 ZFMK Myr3502 Laos, Champasak Province, east of Mekong, Garden of Erawan Riverside Hotel Wesener 2019
16 Z. macula sp. nov. PP754589 CUMZ (M54) Thailand, Sra Kaeo Province, Mueang Sra Kaeo District, Wat Tham Khao Maka This study
17 Z. macula sp. nov. PP754590 CUMZ (M55) Thailand, Sra Kaeo Province, Mueang Sra Kaeo District, Wat Tham Khao Maka This study
18 Z. macula sp. nov. PP754584 CUMZ (M417) Thailand, Chantaburi Province, Khlung District, Thaeo Khlong Khlung Monastery This study
19 Z. macula sp. nov. PP754585 CUMZ (M418) Thailand, Chantaburi Province, Khlung District, Thaeo Khlong Khlung Monastery This study
20 Z. macula sp. nov. PP754586 CUMZ (M421) Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim This study
21 Z. macula sp. nov. PP754587 CUMZ (M422) Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim This study
22 Z. macula sp. nov. PP754588 CUMZ (M424) Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim This study
23 Z. medongensis Zhao & Liu, 2022 OP339793 SCAU XZ01 China, Xizang Autonomous Region, Medog County Zhao et al. 2022
24 Z. ovalis Gray, 1832 JX486068 ZFMK Myr 0832 Vietnam, Dong Nai Province, Cat Tien National Park Golovatch et al. 2012
25 Z. panhai Srisonchai, Sutcharit & Likhitrakarn, 2021 OM509643 ZFMK MYR 8118 Thailand, Ratchaburi Province, Ratchaburi-Photharam Districts Bhansali and Wesener 2022
26 Z. panhai Srisonchai, Sutcharit & Likhitrakarn, 2021 OM509644 MHNG 3A Thailand, Ratchaburi Province, Chom Bueang District, Tham Kao Bin Forest Park Bhansali and Wesener 2022
27 Z. panhai Srisonchai, Sutcharit & Likhitrakarn, 2021 OM509645 ZFMK MYR 8116 Thailand, Ratchaburi Province, Ratchaburi-Photharam Districts Bhansali and Wesener 2022
28 Z. phrain Likhitrakarn & Golovatch, 2021 OM509634 ZFMK MYR 3499 Thailand, Chiang Mai Province, Chiang Dao District, Padeng Lodge Bhansali and Wesener 2022
29 Z. phrain Likhitrakarn & Golovatch, 2021 OM509635 MYR3500 Thailand, Chiangmai Province, Mueang District, Doi Suthep Bhansali and Wesener 2022
30 Z. phrain Likhitrakarn & Golovatch, 2021 OM509636 SMF Thailand, Chiang Mai Province, Chiang Dao District, Tham Houay Luk Bhansali and Wesener 2022
31 Z. phrain Likhitrakarn & Golovatch, 2021 OM509637 SMF Thailand, Chiang Mai Province, Chiang Dao District, Doi Chiang Dao, Ma Lee’s Resort Bhansali and Wesener 2022
32 Z. phrain Likhitrakarn & Golovatch, 2021 OM509638 SMF Thailand, Chiang Mai Province, Chai Prakan District, Tham Ngam Bhansali and Wesener 2022
33 Z. phrain Likhitrakarn & Golovatch, 2021 OM509639 ZFMK MYR 4907 Thailand, Chiang Mai Province, Chiang Dao Bhansali and Wesener 2022
34 Z. phrain Likhitrakarn & Golovatch, 2021 OM509640 MHNG 5G Thailand, Lamphun Province, Mae Tha District, Doi Khuntan National Park Bhansali and Wesener 2022
35 Z. phrain Likhitrakarn & Golovatch, 2021 OM509641 MHNG 5I Thailand, Chiang Mai Province, Chiang Dao District, Doi Chiang Dao Bhansali and Wesener 2022
36 Z. phrain Likhitrakarn & Golovatch, 2021 OM509642 NHMD K35 Thailand, Chiang Mai Province, Ban Musue Bhansali and Wesener 2022
37 Z. siamensis Hirst, 1907 JX486067.2 FMNH-INS-72669 Thailand, Chonburi Province, Sichang District, Koh Sichang Golovatch et al.2012
38 Z. siamensis Hirst, 1907 OR530089 CUMZ Thailand, Chonburi Province, Sichang District, Koh Sichang Srisonchai et al. 2023
39 Z. siamensis Hirst, 1907 PP754592 CUMZ (M455) Thailand, Chachoengsao Province, Phanom Sarakham District, Wat Khao Hin Sorn This study
40 Z. siamensis Hirst, 1907 PP754591 CUMZ (D397) Thailand, Srakaeo Province, Khao Chakan District, Wat Tham Khao Chan This study
41 Zephronia sp. (K45 Aow Noi Temple) MW898741 NHMD K55 Thailand, Prachuap Kiri Khan Province, Mueang District, Aow Noi Temple Rosenmejer et al. 2021
42 Zephronia sp. (Aow Noi Temple) MW898742 ZFMK MYR 8787 Thailand, Prachuap Kiri Khan Province, Mueang District, Aow Noi Temple Rosenmejer et al. 2021
43 Z. viridisoma Rosenmejer & Wesener, 2021 MW898739 NHMD 621695 Thailand, Nakhon Si Thammarat Province, Sichon District, Khao Lark Waterfall Rosenmejer et al. 2021
44 Z. viridisoma Rosenmejer & Wesener, 2021 MW898740 ZFMK MYR 8822 Thailand, Nakhon Si Thammarat Province, Sichon District, Khao Lark Waterfall Rosenmejer et al. 2021
45 Z. zhouae Zhao & Liu, 2022 OP339794 SCAU YN02 China, Yunnan Province, Diqing Tibetan Autonomous Prefecture, Weixi County, Laowo Village Rosenmejer et al. 2021
Outgroups
46 S. aesculus Rosenmejer & Wesener, 2021 MW898737 NHMD 621693 Thailand, Phuket Province, Kathu District, Forest Rosenmejer et al.2021
47 S. cf. aesculus Rosenmejer & Wesener, 2021 MW898738 NHMD 621694 Thailand, Nakhon Si Thammarat Province, Khao Luang NP Rosenmejer et al. 2021
48 S. benquii Liu & Wesener, 2022 OP339792 SCAU MMY01 China, Guizhou, Tongren City, Jiangkou County, Guanhe Town, Guanhe Village, Maomaoyan Zhao et al. 2022
49 S. bolavensis Wesener, 2019 MK330982 MHNG LT-10/24 Laos, Champasak Province, Bolaven Plateau, 3 km S of Ban Nong Luang, Tad Kameud Wesener 2019
50 S. denticulatum Wesener, 2019 MK330984 MHNG LT-10/12 Laos, Oudomxai Province, ca 3 km E of Tad Lak 11, SE of Oudomxai city Wesener 2019
51 S. huzhengkuni Zhao, Yu & Liu, 2020 MT657327 SCAU SP02 China, Guizhou Province, Tongren City, Fanjingshan National Nature Reserve Zhao et al. 2020
52 S. huzhengkuni Zhao, Yu & Liu, 2020 MT657328 SCAU SP03 China, Guizhou Province, Tongren City, Fanjingshan National Nature Reserve Zhao et al. 2020
53 S. lachneeis Wesener, 2019 MK330983 MHNG LT-10/12 Laos, Oudomxai Province, ca 3 km E of Tad Lak 11, SE of Oudomxai city Wesener 2019
54 S. laoticum Wesener, 2019 MK330975 SMF Laos, Vientiane Province, Vang Vieng Wesener 2019
55 S. meridionalis Bhansali & Wesener, 2022 OM509648 MHNG 4B-2 Thailand, Yala Province, Bannang Sata District, Bang Lang National Park, near Than To Waterfall Bhansali and Wesener 2022
56 S. nigrum Wesener, 2019 MK330976 SMF Laos, Champasak Province, Muang Bachieng, Ban Lak 35, Tad Etu Wesener 2019
57 S. peterjaegeri Wesener, 2019 MK330972 SMF SD553 Laos, Luang Prabang Province, SE Luang Prabang, Nam Khan, Ban Pak Bak, Houay Kho Wesener 2019
58 S. phouloei Wesener, 2019 MK330974 ZMUC00040257 Laos, Houaphan Province, Phou Loei Wesener 2019
59 S. schwendingeri Wesener, 2019 MK330978 MHNG LT-10/03 Laos, Vientiane Province, trail to Tham Pou Kham, W. of Vang Vieng Wesener 2019
60 S. schwendingeri Wesener, 2019 MK330981 SMF Laos, Vientiane Province, Vang Vieng, Tham Pou Kham Wesener 2019
61 Sphaerobelum sp. L07 MK330979 ZMUC00040261 Laos, Khammouane Province, Ban Khounkham [Khun Kham] (Nahin) Wesener 2019
62 Sphaerobelum sp. L10 MK330980 SMF Laos, Vientiane Province, Vang Vieng, W. of Nam Song, Tham Nam Or Khem Wesener 2019
63 S. spinatum Wesener, 2019 MK330973 ZMUC00040258 Laos, Vientiane Province, Phou Khao Khouay Wesener 2019
64 S. truncatum Wongthamwanich, 2012 JN885184 FMNH-INS 0000 072 674 Thailand, Nan Province, Song Khwae District, Na Rai Luang Subdistrict, Pang Hi Village Wongthamwanich et al. 2012
65 S. turcosa Srisonchai & Pimvichai, 2023 OR530087 CUMZ-Zeph0012 Thailand, Loei Province, Mueang Loei District, Phu Pha Lom Forest Park Srisonchai et al. 2023
66 S. turcosa Srisonchai & Pimvichai, 2023 OR530087 CUMZ-Zeph0012 Thailand, Loei Province, Mueang Loei District, Phu Pha Lom Forest Park Srisonchai et al., 2023
67 S. tujiaphilum Zhao & Liu, 2022 OP339783 SCAU SD02 China, Guizhou, Tongren City, Jiangkou County, Guanhe Town, Sidu Village Zhao et al. 2022
68 S. tujiaphilum Zhao & Liu, 2022 OP339784 SCAU SD01 China, Guizhou, Tongren City, Jiangkou County, Guanhe Town, Sidu Village Zhao et al. 2022
69 S. tujiaphilum Zhao & Liu, 2022 OP339785 SCAU BHS01 China, Guizhou, Tongren City, Jiangkou County, Taiping Town, Baiheshan Village Zhao et al. 2022
70 S. tujiaphilum Zhao & Liu, 2022 OP339786 SCAU JXT01 China, Guizhou, Tongren City, Jiangkou County, Taiping Town, Jiang-xitun Village Zhao et al. 2022
71 S. tujiaphilum Zhao & Liu, 2022 OP339787 SCAU JXT02 China, Guizhou, Tongren City, Jiangkou County, Taiping Town, Jiang-xitun Village Zhao et al. 2022
72 S. tujiaphilum Zhao & Liu, 2022 OP339788 SCAU SD03 China, Guizhou, Tongren City, Jiangkou County, Guanhe Town, Sidu Village Zhao et al. 2022
73 S. tujiaphilum Zhao & Liu, 2022 OP339789 SCAU DW01 China, Guizhou, Tongren City, Jiangkou County, Dewang Town, Xiaobang Village Zhao et al. 2022
74 Arthrosphaera brandtii (Humbert, 1865) FJ409915 FMNH-INS 8650 Tanzania, Usambara hills Wesener et al. 2010
75 Cryxus ovalis (Linnaeus, 1758) JX486069.2 ZFMK MYR0824 Vietnam, Dong Nai Province, Cat Tien National Park Golovatch et al. 2012
76 Epicyliosoma sp. AF218270 NA NA Edgecombe and Giribet 2004
77 Glomeris marginata (Villers, 1789) FJ409909 ZFMK Myr009 Germany, Bonn, Venusberg Wesener et al. 2010
78 Prionobelum inthanonense Donworth & Wesener, 2024 PP297645 MHNG 4E-2 Thailand, Chiang Mai Province, Chom Thong District, Doi Inthanon National Park Donworth and Wesener 2024
79 Prionobelum inthanonense Donworth & Wesener, 2024 PP297646 MHNG 7A Thailand, Chiang Mai Province, Chom Thong District, Doi Inthanon National Park Donworth and Wesener 2024
80 Prionobelum naevium Donworth & Wesener, 2024 PP297647 MHNG 4B-1 Thailand, Yala Province, Than To District, Bang Lang National Park, Than To Waterfall Donworth and Wesener 2024
81 Prionobelum naevium Donworth & Wesener, 2024 PP297648 NHMD 1184671 Thailand, Yala Province, Than To District, Bang Lang National Park Donworth and Wesener 2024
82 Prionobelum naevium Donworth & Wesener, 2024 PP297649 NHMD 1184672 Thailand, Yala Province, Than To District, Bang Lang National Park Donworth and Wesener 2024
83 Prionobelum naevium Donworth & Wesener, 2024 PP297650 NHMD 1184673 Thailand, Yala Province, Than To District, Bang Lang National Park Donworth and Wesener 2024
84 Sphaeromimus splendidus Wesener & Sierwald, 2005 FJ409917 FMMC-INS 6702 Madagascar, Sainte Luce S9 Wesener et al. 2010

The inspection of sequencing chromatograms was performed in MEGA 7 (Kumar et al. 2016) in order to check for missing sites. The stop codons and misaligned regions were also carefully checked by aligning all sequences into an appropriate reading frame with a few nucleotide sequences removed at the 5' end and translated to amino acids. All sequences were aligned in MEGA 7 and the aligned data were analysed using JModelTest2 on XSDXE 2.1.6 (Darriba et al. 2012) through the CIPRES Gateway (Miller et al. 2010) to test nucleotide evolution models and to infer the best-fit substitution model for the data.

The phylogenetic trees were generated under two approaches: maximum likelihood (ML) and Bayesian inference (BI). For ML, the analysis was conducted using IQ-tree on XSEDE 1.6.6 (Minh et al. 2020) via the CIPRES Gateway (Miller et al. 2010) with the GTR+I+G model and with 10,000 bootstrap replicates. For BI, we used KAKUSAN 4.0 (Tanabe 2011) to prepare the original file with the adjustments of the best-fit model (GTR+I+G). The analysis was conducted in MrBayes on XSEDE 3.2.7a (Ronquist et al. 2012) via the CIPRES Gateway with Markov Chain Monte Carlo algorithms (MCMC) and with a random starting tree, running for 50 million generations, sampling every 1,000 generations. The ML and BI trees were visualized in FigTree v. 1.4.0 (Rambaut 2010) and then visually processed in Adobe Illustrator 2021. To evaluate node robustness, ML bootstrap support values (BS) > 70% and BI posterior probabilities (PP) > 0.95% are interpreted as strong support (Huelsenbeck and Hillis 1993; Larget and Simon 1999). We also evaluated the intergeneric, interspecific, and intraspecific genetic distances using uncorrected p-distance in MEGA 7 (Kumar et al. 2016) under the pairwise deletion parameter.

Results

Genetic analyses

The final aligned COI sequences were composed of 658 base pairs, and the sequence annotation contained 375 variable sites, 263 conservative sites and 312 sites were parsimony informative. The percentual distances between outgroups (Arthrosphaera brandtii + Epicyliosoma sp. + Arthrosphaera brandti + Sphaeromimus splendidus) and Zephronia + Sphaerobelum + Prionobelum + Cryxus were 25.11–46.39%. The intergeneric distances between Zephronia and Sphaerobelum ranged between 7.19 to 31.62%, between 19.13 to 28.63% for Zephronia and Prionobelum, and between 21.19 to 28.75% for Zephronia and Cryxus. The interspecific distances within Zephronia, Sphaerobelum, Prionobelum each ranged from 9.91–29.93%, 10.86–31.21% and 20.60–22.12%, respectively. The intraspecific distances of Z. chantaburiensis sp. nov. and Z. macula sp. nov. ranged between 0–0.15% and 0.15–4.40%, respectively. The two new species were separated from each other by an interspecific p-distance of 18.94–19.71%. Information regarding genetic distances is presented in the Suppl. material 1.

BI and ML methods returned different topologies, especially at deep nodes (Fig. 1 and Suppl. material 2). However, the relationships at younger internal nodes and terminal nodes of most species were still similar to each other, especially for Zephronia sensu stricto (s.s.). The tree topology based on BI compared to ML is presented as an overview to interpret the results (Fig. 1).

Figure 1. 

Phylogenetic tree based on Bayesian Inferences (BI). Numbers at nodes indicate Bayesian posterior probabilities (PP)/ bootstrap support (BS). The dash “-” at the nodes refer to a different topology in the BI analysis compared to ML (see Supplemental Material 2). Colours denote the two new species (Zephronia chantaburiensis sp. nov. and Zephronia macula sp. nov.), corresponding to the live photograph. Scale bar represents substitutions/site.

The low support values of deep nodes in our trees based on the COI gene alone do not allow for a conclusion on the relationships among the species of Zephronia as well as of those in Sphaerobelum, Prionobelum, and Cryxus. The preliminary data, however, questions the monophyly of these genera.

According to the results, the trees from both BI and ML clearly show that the genus Zephronia is recovered as polyphyletic, with the clades formed by Z. viridisoma and Zephronia sp. (Aow Noi Temple) as a well-supported sister group to all other species of Zephronia and Sphaerobelum located in a trichotomy with a branch supporting Prionobelum and Cryxus Leach, 1814 (PP = 1.0, BS = 98). Z. hui and Z. phrain cluster among species of Sphaerobelum, but with low statistical support. Morphologically aberrant species of Sphaerobelum cluster with Prionobelum inthanonense. All other Thai, Laotian and Cambodian Zephronia s.s. are in a well-supported monophylum in both BI and ML (PP = 0.97, BS = 82) including the two new species described here (Fig. 1). Regarding the new species, each is recovered as monophyletic with strong support (BS = 100, PP = 1.0) and the two are grouped together with members of the Zephronia s.s. species group (Z. dawydoffi, Z. laotica, Z. ovalis, and Z. siamensis).

Systematics

Family Zephroniidae Gray, 1843

Subfamily Zephroniinae Gray, 1843

Tribe Zephroniini Gray, 1843

Zephronia Gray, 1832

Differential diagnosis

Differs from all other genera of Zephroniinae by the combination of the following characters: 1) Body length 18–50 mm. 2) Antennae flattened laterally, usually axe-shaped, with numerous (> 4) apical cones. 3) Endotergum (tergite underside) weakly modified: posterior margin (pm) usually flat; outer area (os) without setae; marginal bristles arranged in 1–5 rows; middle area (ma) often with a single row of circular cuticular impressions. 4) Tarsi of legs 5–21 often with more than one apical spine (with very few species with only 1) and several ventral spines. 5) Anterior telopods with four podomeres distal to syncoxite; telopoditomere 2 with a large, curved process forming a clamp-like; telopoditomeres 3 and 4 simple. 6) Posterior telopods with four telopoditomeres; immovable finger (process of telopoditomere 2 slender, apically curved; telopoditomeres 3 and 4 with two membranous lobes; telopoditomere 3 also with a row of conspicuous, crenulated teeth, larger than elopoditomere 4; telopoditomere 4 with few (one or two) sclerotized spines. 7) Female vulvae conspicuous, slender; operculum usually round.

Zephronia is one of the most species-rich genera of the family with more than 50 species currently placed in the genus. Numerous species currently placed in the genus are in need of a revision, some only known from the female, and might be placed in separate genera in the future. The posterior telopod in Zephronia consists of four podomeres, unlike Castanotherium Pocock, 1895 whose species have three podomeres only. Podomere 4 of the posterior telopod in Zephronia is not strongly curved or overlapping the process of podomere 2 as in Cryxus Leach, 1814. The process of podomere 2 in Zephronia is never apically enlarged or swollen like in Sphaerobelum Verhoeff, 1924. The osterior telopod on podomere 3 in Zephronia has crenulated teeth, unlike Kophosphaera Attems, 1935 which lacks sclerotized teeth or spines entirely. The posterior telopod of Zephronia species is identical to those of species of Sphaeropoeus Brandt, 1833, Prionobelum Verhoeff, 1924, Tigridosphaera Jeekel, 2000, and Indosphaera Attems, 1935. Zephronia species differ from species of Sphaeropoeus and Prionobelum in the anterior telopods, lacking the characteristic processes on joints three (Sphaeropoeus) and four (Prionobelum). Zephronia species differ from Indosphaera in the coxae of leg 2 in females being separate, not fused. Zephronia species are currently impossible to distinguish from species of Tigridosphaera as that species-poor genus is in need of revision. Females of Zephronia differ in their shape of the operculum of the vulva from species of Indosphaera, where it is much lower than in Zephronia species, as well as from species of Sphaeropoeus and Prionobelum where the operculum is greatly enlarged and almost rectangular in shape.

Zephronia s.s

Differing from all Zephronia s.l. by the position of the organ of Tömösváry, which is located at the brim and not inside the antennal groove as in all other known Sphaerotheriida. Zephronia s.s. includes the type species of the genus, Z. ovalis, as well as Z. chantaburiensis sp. nov., Z. chrysomallos, Z. dawydoffi, Z. erawani, Z. enghoffi, Z. golovatchi, Z. hui Liu & Wesener, 2022, Z. konkakinhensis Semenyuk, Golovatch & Wesener, 2018, Z. lannaensis, Z. laotica, Z. macula sp. nov., Z. medongensis Zhao & Liu, 2022, Z. montis Semenyuk, Golovatch & Wesener, 2018, Z. panhai, Z. siamensis and Z. zhouae Zhao & Liu, 2022. See more details for the genus in Wesener (2016) and Semenyuk et al. (2018), and a recent update of Thai species in Bhansali and Wesener (2022).

List of 11 Zephronia species occurring in Thailand

  1. Z. chantaburiensis Srisonchai & Wesener, sp. nov.
  2. Z. chrysomallos Bhansali & Wesener, 2022
  3. Z. enghoffi Srisonchai & Likhitrakarn, 2021
  4. Z. erawani Bhansali & Wesener, 2022
  5. Z. golovatchi Srisonchai & Likhitrakarn, 2021
  6. Z. lannaensis Likhitrakarn & Golovatch, 2021
  7. Z. macula Srisonchai & Wesener, sp. nov.
  8. Z. panhai Srisonchai & Likhitrakarn, 2021
  9. Z. phrain Likhitrakarn & Golovatch, 2021
  10. Z. siamensis Hirst, 1907
  11. Z. viridisoma Rosenmejer & Wesener, 2021

Zephronia chantaburiensis Srisonchai & Wesener, sp. nov.

Figs 2, 3, 4, 5, 6, 7, 14A–D, 15

Type specimens

Holotype • ♂ (CUMZ-MYR0013); Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim (Khao Sukim Temple); 12°45'47"N, 102°01'56"E; ca 53 m a.s.l.; 14 June 2023; leg. R. Srisonchai and KKUMZ students. Paratypes. • 29 ♂, 16 ♀ (CUMZ-MYR0014), same data as holotype; • 2 ♂, 1 ♀ (NHMD1184695), same data as holotype; • 1 ♂, 1 ♀ (NHMW10436), same data as holotype; • 1 ♂, 1 ♀ (ZFMK-MYR13659), same data as holotype.

Additional material

• 26 juveniles (CUMZ), same data as holotype.

Diagnosis

The position of the organ of Tömösváry at the brim and not inside the antennal groove (Fig. 3B) identifies this species as a member of the Zephronia s.s. species group (see Semenyuk et al. 2018), with which it also aligns genetically (Fig. 1). This small brown species (body length ca 20 mm) with short golden hair (Fig. 2A–F) differs from all other Zephronia s.s. species, except for Z. macula sp. nov. found in direct sympatry, described below, in the presence of only a single apical spine on the tarsus of legs 4–21 (2 or 3 in the other species). Midbody endotergum with one row of marginal bristles with longest bristles reaching up to posterior margin, immovable finger (process) of telopoditomere 2 on anterior telopod relatively long and slender. Similar in these respects only to Z. macula sp. nov. but differs from the latter species by the tergite coloration lacking dark or greenish-dark colour spots, the operculum of the female being more slender, the femur of the walking legs being slightly wider than long (slightly longer than wide in Z. macula sp. nov.), and the female subanal plate having a strongly concave margin. Genetically distant from other species by 18.94–26.82% p-distance in the COI barcoding fragment.

Figure 2. 

Photograph of live specimens of Zephronia chantaburiensis sp. nov. and habitats A–F paratypes (CUMZ-MYR0014) G coexisting species (Z. macula sp. nov.) H granite habitat at the type locality. Scale bars: 0.5 mm.

Figure 3. 

Zephronia chantaburiensis sp. nov., ♂ paratype (CUMZ-MYR0014) — SEM A head, collum and antenna, dorsal view B ommatidia, inset: organ of Tömösváry C antenna, anterior view D antennal disc, anterior view E apical cones, lateral view F, I gnathochilarium, posterior and ventral views, respectively G sensory cones on central pad, ventral view H sensory cones on inner palpi, black and white arrows point to different types of cones, ventral view J epipharynx, ventral view K mandible, mesal view. Abbreviations: 3it = 3-combed inner tooth, co = condylus, Cp = central pad, ct = central tooth, et = external tooth, Et = external tooth, Ia = inner area, il = incisura lateralis, ip = inner palpi, ll = lamellae linguales, me = mentum, mp = molar plate, pl = pectinate lamellae, rsp = row of spines, sc = sensory cone, st = stipite. Scale bars in millimeters.

Description

Measurements : Male holotype. Body length 20 mm. Width, of thoracic shield 9 mm, of tergite 7 = 10 mm (= broadest). Height of tergite 7 = 7 mm (= highest). Males: body length = 19–23 mm. Width, of thoracic shield = 8–9 mm, of tergite 7 = 9–10 mm. Height of tergite 7 = 6–8 mm. Females: body length = 19–22 mm. Width, of thoracic shield = 8–9 mm, of tergite 7 = 9–11 mm. Height of tergite 7 = 6–8 mm (= highest).

Colouration (Fig. 2A–F): In life with body of brown colour. Head, collum, thoracic shield and tergites brown. Antenna, legs, and venter light brown. Anal shield dark brown (rarely brown), except anterior and posterior margins pale brown. Tergites dorsally with dark brown stripe, clearly seen when rolled up. Specimens in alcohol after one year changed to pale brown.

Head (Fig. 3A, B): Trapeziform, densely setose; anterior part with setae longer than in posterior part; each seta located inside small pit. With 57–65 ommatidia (ocelli) in males and 60–65 in females. Aberrant ommatidium located at brim of antennal groove. Organ of Tömösváry situated near the base of antenna, separated from eye field. No sclerotized crest/ridge between antennal socket and eye field.

Antennae (Figs 2B, C, 3A, C–E): Short and stout, covered by long and dense setae; last antennomere reaching back to leg pair 2 or 3. Lengths of antennomeres: 1=2=3=4<5<<6. Antennomere 6 slightly flattened apically, axe-shaped; apically with sensilla basiconica. Apical disc flat; with 30–41 (male) or 25–35 (female) apical cones.

Epipharynx (Fig. 3J): With a regular central tooth (ct); inner tooth conspicuous and flat; laterally with numerous long external teeth (et); inner area with a single row of fringed spines (rsp) on each side.

Gnathochilarium (Fig. 3F–I): Structure as typical as for Sphaerotheriida. Lamellae linguales (ll) oval, slightly concave apically, with long setae. Central pads (Cp) modified, with numerous “pillows” of sensory cones (sc) (Fig. 3H); two types of sensory cones (one with a pillow and another without a pillow). Stipites (st) large, densely setose; located laterally to lamellae linguales. Mentum (me) large, fused, with sparse and long setae. Lateral palpi inconspicuous. Inner palpi (ip) with sensory cones (sc) arranged in single field (Fig. 3G).

Mandibles (gnathal lobe) (Fig. 3K): With undivided external tooth (Et) and with conspicuous 3-combed inner tooth (3it). With 5–7 pectinate lamellae (pl). Inner area (Ia) with group of long and tiny teeth. Molar plate (mp) flat, velvet-like; lacking a membranous fringe. Condylus (co) conspicuous, apically with one distinct ridge.

Tegument (Figs 2A–F, 4): Quite dull; collum, thoracic shield, tergite and anal shield densely covered by tiny golden setae; each seta located in a pit. Anterior margins of midbody tergite and of anal shield with lower number of setae than posterior margins. Posterior rim in dorsal and ventral side of anal shield with a few small setae.

Figure 4. 

Zephronia chantaburiensis sp. nov., ♂ paratype (CUMZ-MYR0014) — SEM A surface of collum B, C thoracic shield, all in dorsal views (anterior margin and surface, respectively) D–G midbody tergite, all in dorsal views (overview, anterior margin, surface, and posterior margin, respectively) H–J anal shield, all in dorsal views (overview, anterior margin and cuticular impressions, respectively) K–M anal shield, underside, all in ventral views (surface, posterior margin, and overview, respectively). Scale bars in millimetres.

Collum (Figs 2B, 3A): Subsemicircular; tip of lateral margin obtuse, densely setose.

Thoracic shield (Fig. 4B): With shallow and large groove separated by a large and long ridge. Slope towards groove without keel. Groove and ridge smooth, without setae.

Midbody tergite (Fig. 4D–G): With a row of oval impressions and numerous tiny tubercles at anterior edge. Inner area with crenate barrier, forming a wide and shallow groove. Tips of midbody paratergites projecting caudoventrad.

Anal shield (Fig. 4H–M): Slightly sexually dimorphic, in female large and well-rounded, in male slightly slenderer. With a row of oval impressions and several tiny tubercles at anterior edge. Inner area with crenate barrier, forming a wide and shallow groove. Underside with a single, short, black locking carina; twice as long as those of tergites, as long as half of tarsus.

Endotergum of thoracic shield and midbody tergite (Fig. 14A–D): Thoracic shield similar to midbody tergite. Posterior margin (pm) flat, regular. Outer area (os) without setae. Marginal bristles arranged in one row, tip of the longest bristles very slightly protruding above posterior margin. Middle area (ma) with a single row of conspicuous, circular cuticular impressions; distance between impressions twice as long as individual diameter. Inner area (ia) without tubercles or setae.

Pleurite (laterotergite) (Fig. 5B): With short setae. First pleurite slender, boomerang-shaped; apical margin slightly attenuated; strongly projecting into a wide tip. Pleurite 2 wider than first one, projecting into obtuse tip. The remaining pleurites flat and wide, lamella-like, apical margin slightly extended.

Figure 5. 

Zephronia chantaburiensis sp. nov. SEM. A–G, L ♂ paratype (CUMZ-MYR0014) J, K ♀ paratype (CUMZ-MYR0014) A collum B first pleurite C subanal plate D legs 1 E stigmatic plate 1 F legs 2 with gonopores G leg 10 (arrow points to femoral ridge) H coxal process on leg 10 I small teeth on femur J leg 2 with vulvae K vulvae L tarsus 10. Abbreviation: bu = bursa, cx = coxa, fe = femur, op = operculum, po = postfemur, pre = prefemur, st-pl = stigmatic plate, ta = tarsus, ti = tibia. Scale bars in millimetres.

Subanal plate of female (Fig. 5C): Large and wide, semicircular; sparsely setose; apical margin strongly concave.

Stigmatic plates (Fig. 5D, E): First stigmatic plate slender, apex well-rounded, slightly curving towards coxa (Fig. 5E). Second stigmatic plate in both sexes without any curve (Fig. 5F, J).

Legs (Fig. 5D–L): All podomeres densely setose. Coxa (cx) large; coxal process absent in first and second legs; legs 3–21 marginally with large and dentate process. Prefemur (pre) short and stout; apico-mesally with a weak projection; mesal margin with small spines. Femur (fe) stout; with long ridge in all leg-pairs; apico-mesally massively enlarged with a strong projection carrying dentate margin (conspicuous teeth on margin). Postfemur (po) and tibia (ti) short. Tarsus (ta) of midbody legs quite long and slender; as long as length of prefemur+femur combined; first two leg-pairs without an apical spine; leg-pair 1 with 1 ventral spine; leg-pair 2 with 3 ventral spines; leg-pair 3 with 5 or 6 ventral spines and 1 apical spine; leg pairs 4 with 7 or 8 ventral spines and 1 apical spine; leg pairs 5–21 with 7–9 ventral spines and 1 apical spine. In leg 9, femur slightly wider than long (1.1×), tarsus 4× longer than wide. Claw normal, with a small notch at base.

Male sexual characters (Fig. 5F): Gonopore large, covered by long setae; with divided sclerotized plates, triangular.

Anterior telopods (Fig. 6A, B, E–G): First telopoditomere rectangular. Telopoditomere 2 large, as long as telopoditomere 3. Immovable finger (process of telopoditomere 2) long and slender; almost as long as movable finger (= combination of telopoditomeres 3+4); clearly seen in posterior and anterior views; strongly curved; tip obtuse, directed anteriad; with a membranous lobe and sclerotized spots located at inner margin. Telopoditomere 3 as long as telopoditomere 4, clearly demarcated from telopoditomere 4 by conspicuous suture; apically with few crenulated teeth (cr-t). Tepoloditomere 4 apically with large and long setae.

Posterior telopods (Figs 6A–D, H–J; 7): Consisting of 4 telopoditomeres. First telopoditomere rectangular, slightly longer than wide. Telopoditomere 2 large and stout. Immovable finger (process of telopoditomere 2) long and slender, 3× longer than wide, with a characteristic swelling mesally; slightly longer than movable finger (= combination of telopoditomeres 3+4); attenuate near tip; tip obtuse; tip in situ curving anteriad; inner margin with few conspicuous sclerotized spots (scl-s) and two membranous lobes (ml). Telopoditomere 3 long, 2.5× longer than wide, with a membranous ledge and single spine at excavate inner margin. Telopoditomere 4 relatively short; 4× shorter than telopoditomere 3; 1.5× longer than wide, slightly tapering toward apex; with two long spines located on membranous ledge; posteriorly with 9–12 crenulated teeth (cr-t). Telopoditomeres 1 and 2 in anterior view mostly covered by setae, in posterior view mostly glabrous. Telopoditomeres 3 and 4 glabrous, except small area at basal part of telopoditomere 3 with setae. Inner horns with sharp-edged tips, slightly curved caudad.

Figure 6. 

Zephronia chantaburiensis sp. nov., ♂ paratype (CUMZ-MYR0014) — SEM A–D overview of telopods (anterior view, sublateral view with emphasis on anterior telopod, posterior view and ventral view, respectively) E–G anterior telopod (telopoditomeres 2–4, immovable finger and crenulated teeth, respectively) H–J posterior telopod, all in sublateral views (sclerotize spots on immovable finger, telopoditomeres 2–4 with emphasis on membranous lobe and crenulated teeth, respectively). Abbreviations: cr-t = crenulated teeth, ml = membranous lobe, scl-s = sclerotized spots. Scale bars in millimeters.

Figure 7. 

Zephronia chantaburiensis sp. nov., ♂ paratype (ZFMK-MYR13659), drawings — right posterior telopod A anterior view B posterior view. Abbreviations: cr-t = crenulated teeth, imf = immovable finger, ml = membranous lobe. Scale bar: 0.5 millimeters micrometres.

Female sexual characters (Fig. 5J, K): Vulva large, slender, sparsely setose; covering almost 1/2 of coxa; located at mesal margin; extending mesally to base of prefemur. Operculum (op) round, mesal margin protruding into an oval-like lobe, tip of operculum obtuse. Bursa (bu) large, demarcated from operculum by a triangular groove.

Remarks

Negligible colour variation is observed in the anal shield between different living specimens. Generally, they are dark brown in most specimens and pale brown in others. Females are typically of the same size as males.

Distribution and habitat

Zephronia chantaburiensis sp. nov. is currently known only from the type locality in Chantaburi Province and is therefore here regarded as an endemic species. The new species was encountered during the day time in evergreen forest in granitic habitat (Fig. 2H). They hide themselves beneath thick leaf litter and decayed wood logs (Fig. 2G). Co-occurring and dominant millipedes are included, Z. macula sp. nov. and Desmoxytes euros Srisonchai et al., 2018.

Etymology

The name is an adjective referring to the province (Chantaburi) where the type locality is located.

Zephronia macula Srisonchai & Wesener, sp. nov.

Figs 8, 9, 10, 11, 12, 13, 14E–H, 15

Type specimen

Holotype • ♂ (CUMZ-MYR0015); Thailand, Chantaburi Province, Kaeng Hang Maeo District, near Khao Wong Kot Cave, Thamma Sooksawang Temple; 12°53'51.7"N, 101°48'59.7"E; ca. 60 m a.s.l.; 14 June 2023; leg. R. Srisonchai and KKUMZ students. Paratypes. • 53 ♂, 74 ♀ (CUMZ-MYR0016), same data as holotype; • 2 ♂, 2 ♀ (NHMD1184696), same data as holotype; • 2 ♂, 2 ♀ (NHMW10437), same data as holotype; • 2 ♂, 2 ♀ (ZFMK-MYR13660), same data as holotype.

Additional material

• 29 juveniles (CUMZ); Thailand, Chantaburi Province, Kaeng Hang Maeo District, near Khao Wong Kot Cave, Thamma Sooksawang Temple; 12°53'51.7"N, 101°48'59.7"E; ca. 60 m a.s.l.; 14 June 2023; leg. R. Srisonchai and KKUMZ students; • 26 ♂, 31 ♀ (CUMZ); Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim (Khao Sukim Temple); 12°45'47"N, 102°01'56"E; ca. 53 m a.s.l.; 14 June 2023; leg. R. Srisonchai and KKUMZ students; • 18 ♂, 26 ♀ (CUMZ); Thailand, Chantaburi Province, Khlung District, Thaeo Klong Khlung Monastery; 12°28'53.3"N, 102°13'06.3"E; ca. 74 m a.s.l.; 14 June 2023; leg. R. Srisonchai and KKUMZ students; • 26 ♂, 65 ♀; 47 juveniles (CUMZ); Thailand, Chantaburi Province, Makham District, Wat Khao Banchob, 12°51'09.0"N, 102°12'15.0"E; ca. 110 m a.s.l.; 13 June 2023; leg. R. Srisonchai and KKUMZ students • 33 ♂, 43 ♀ (CUMZ); Thailand, Chonburi Province, Bo Thong District, Wat Khao Yai Aran Khiri; 13°14'54.7"N, 101°37'29.6"E; ca. 160 m a.s.l.; 15 June 2023; leg. R. Srisonchai and KKUMZ students; • 66 ♂, 25 ♀ (CUMZ); Thailand, Rayong Province, Makham District, Wat Pa Theprangsi (= Wat Khao Hin Tang); 12°42'03.3"N, 101°32'17.3"E; ca. 80 m a.s.l.; 14 June 2023; leg. R. Srisonchai and KKUMZ students; • 5 ♂, 7 juveniles (CUMZ); Thailand, Sa Kaeo Province, Khlong Hat District, Phet Pho Thong Cave; 13°24'53.5"N, 102°19'35.1"E; ca. 245 m a.s.l.; 12 June 2023; leg. R. Srisonchai and KKUMZ students; • 1 ♂, 2 ♀ (CUMZ); Thailand, Sra Kaeo Province, Mueang Sra Kaeo District, Wat Tham Khao Maka; 13°47'11.9"N, 101°56'51.8"E; ca. 70 m a.s.l.; August 2014; leg. ASRU members

Diagnosis

The position of the organ of Tömösváry in this small Zephronia with an axe-shaped antennomere 6 identifies Z. macula sp. nov. as a member of the Zephronia s.s. species-group (Semenyuk et al. 2018). Z. macula sp. nov. differs from all other known species of the group except for the syntopic Z. chantaburiensis sp. nov., in the presence of only a single apical spine on the tarsi of legs 4–21 (at least 2 or 3 in the other Zephronia). Z. macula sp. nov. differs from Z. chantaburiensis sp. nov. by having a combination of distinct characters, viz. tergite with dark or greenish dark spots, femur of walking legs less strongly widened, slightly longer than wide (wider than long in Z. chantaburiensis sp. nov.), bristles of midbody endotergum reaching to posterior margin, operculum of female vulva not protruding and round, and immovable finger (process) of telopoditomere 2 of the anterior telopod more curved and much longer. Genetically distant from others by a p-distance of the COI barcoding fragment of 17.93–25.13%.

Description

Measurements : Male holotype. Body length 18 mm. Width, of thoracic shield 8 mm, of tergite 7 = 9 mm (= broadest). Height of tergite 7 = 6 mm (= highest). Males: body length = 18–20 mm. Width, of thoracic shield = 8–9 mm, of tergite 7 = 8–10 mm. Height of tergite 7 = 6–7 mm. Females: body length = 18–20 mm. Width, of thoracic shield = 8–9 mm, of tergite 7 = 9–10 mm. Height of tergite 7 = 6–8 mm (= highest).

Colouration (Fig. 8A–G): Specimen in life with body of brown/greenish brown/reddish brown colour, tergite with dark or greenish dark spots. Head, antenna, and collum dark brown. Thoracic shield, tergites and anal shield brown. Colour in alcohol after one year faded to brown.

Figure 8. 

Photograph of live specimens of Zephronia macula sp. nov. and habitats A–G paratypes (CUMZ-MYR00016) H habitat at Thaeo Khlong Khlung monastery (granitic rocks) I habitat at Phet Pho Thong cave (limestone). Scale bars: 0.5 mm.

Head (Fig. 9A, B): Trapeziform, densely setose; each seta located inside small pit. With 55–65 ommatidia (ocelli) in males and 61–70 in females. At rim of antennal groove with aberrant ommatidium. Organ of Tömösváry located near base of antenna, clearly separated from eye field. No sclerotized crest/ridge between antennal socket and eye field.

Figure 9. 

Zephronia macula sp. nov., ♂ paratype (CUMZ-MYR0016) — SEM A head, collum and antenna, dorsal view B ommatidia, inset: organ of Tömösváry C antenna, anterior view D antennal disc, anterior view E apical cones, lateral view F, I gnathochilarium, posterior and ventral views, respectively G sensory cones on central pad, ventral view H sensory cones on inner palpi, black and white arrows point to different types of cones, ventral view J epipharynx, ventral view, K mandible, mesal view. Abbreviations: 3it = 3-combed inner tooth, co = condylus, Cp = central pad, ct = central tooth, et = external tooth, Et = external tooth, Ia = inner area, il = incisura lateralis, ip = inner palpi, ll = lamellae linguales, me = mentum, mp = molar plate, pl = pectinate lamellae, rsp = row of spines, sc = sensory cone, st = stipites. Scale bars in millimetres.

Antennae (Figs 8C, D, 9A, C–E): Short and stout, covered by long and dense setae; last antennomere reaching back to leg pair 2 or 3 when stretched ventrally. Lengths of antennomeres: 1=2=3=4<5<<6. Antennomere 6 strongly flattened apically, axe-shaped; apically with sensilla basiconica. Apical disc slightly concave, with 48–57 apical cones (male) or 36–43 (female).

Epipharynx (Fig. 9J): With a large central tooth (ct); inner tooth conspicuous and swollen; laterally with group of long external teeth (et); inner area with a single row of fringed spines (rsp) on each side.

Gnathochilarium (Fig. 9F–I): As in Z. chantaburiensis sp. nov. Lamellae linguales (ll) oval, apically concave, with long setae. Central pads (Cp) modified, with numerous “pillows” of sensory cones (sc) (Fig. 9H); two different types of sensory cones (one with a pillow and another without a pillow). Stipites (st) large and stout, densely setose. Mentum (me) large and broad, fused, with long setae. Lateral palpi inconspicuous. Inner palpi (ip) with sensory cones (sc) arranged in single field (Fig. 9G).

Mandibles (gnathal lobe) (Fig. 9K): With undivided external tooth (Et) and with prominent 3-combed inner tooth (3it). With 5–6 pectinate lamellae (pl). Inner area (Ia) with group of long and tiny teeth, spine-like. Molar plate (mp) flat, velvet-like; lacking a membranous fringe. Condylus (co) conspicuous, apically with 2 distinct ridges.

Tegument (Figs 8A–G, 10): Dull; collum, thoracic shield, tergite and anal shield with tiny golden setae; each seta located in small pit. Anterior margins of midbody tergite and of anal shield with lower number of setae than posterior margins. Posterior rim on dorsal and ventral side of anal shield with a few small setae.

Figure 10. 

Zephronia macula sp. nov., ♂ paratype (CUMZ-MYR0016) — SEM A surface of collum B, C thoracic shield, all in dorsal views (anterior margin and surface, respectively) D–G midbody tergite, all in dorsal views (overview, anterior margin, surface, and posterior margin, respectively) H–J anal shield, all in dorsal views (overview, anterior margin and cuticular impressions, respectively) K–M anal shield, underside, all in ventral views (surface, posterior margin, and overview, respectively). Scale bars in millimetres.

Collum (Figs 8C, D, 11A): Sub-semicircular; tip of lateral margin obtuse, covered with fine setae.

Figure 11. 

Zephronia macula sp. nov. — SEM A–G, L ♂ paratype (CUMZ-MYR0016) J, K ♀ paratype (CUMZ-MYR0015) A collum B seventh pleurite C subanal plate D legs 1 E stigmatic plate 1 F legs 2 with gonopores G leg 10 (arrow points to femoral ridge) H coxal process on leg 10 I small teeth on femur J leg 2 with vulvae K vulvae L tarsus 10. Abbreviation: bu = bursa, cx = coxa, fe = femur, op = operculum, po = postfemur, pre = prefemur, st-pl = stigmatic plate, ta = tarsus, ti = tibia. Scale bars in millimetres.

Thoracic shield (Fig. 10B): With a shallow, large, wide groove separated by a large and long ridge. Slope towards groove without keels. Groove smooth, without setae; on a ridge with a few setae.

Midbody tergite (Fig. 10D–G): With a row of oval impressions; anterior margin with tiny tubercles. Inner area with a crenate barrier; a wide and shallow groove. Tips of midbody paratergites projecting ventro-posteriad.

Anal shield (Fig. 10H–M): In female large and well-rounded, in male slenderer. With a row of oval impressions, edge of anterior margin with numerous tiny tubercles (sometimes arranged into rows). Inner area with a row of oval impressions, forming a crenate barrier; with a wide and shallow groove.

Endotergum of thoracic shield and midbody tergite (Fig. 14E–H): Thoracic shield similar to midbody tergites. Overall surface smooth. Posterior margin (pm) flat, not modified. Outer area (os) without setae. Bristles arranged in one row; tip of longest bristles reaching to posterior margin (Fig. 14G, H showing short bristles). Middle area (ma) with a single row of conspicuous, oval cuticular impressions; distance between impressions twice as long as individual diameter. Inner area (ia) without tubercles or setae.

Pleurite (laterotergite) (Fig. 11B): Densely setose. First pleurite boomerang-shaped; apical margin attenuated; strongly projecting into sharp tip. Pleurite 2 projecting into an obtuse tip. Remaining pleurites flat and wide, lamella-like, apical margin extended.

Subanal plate of female (Fig. 11C): Sparsely setose. Large and broad (sometimes quite narrow), semicircular; apical margin slightly concave (rarely truncate/obtuse).

Stigmatic plates (Fig. 11D, E): First stigmatic plate large, slender; apex well-rounded, slightly curved towards basal part of prefemur. Second stigmatic plates in both male (Fig. 11F) and female (Fig. 11J) only very weakly curved towards coxa.

Legs (Fig. 11D–L): All podomeres with long setae. Coxa (cx) large, as long as length of prefemur; coxal process absent in first and second legs; legs 3–21 marginally with dentate process (tooth), conspicuous, broad. Prefemur (pre) stout; apico-mesally with weak projection; mesal margin with conspicuous and small spines. Femur (fe) short and stout, as long as length of prefemur; with a long ridge in all leg-pairs; apico-mesally with strong spines, forming a dentate margin. Postfemur (po) and tibia (ti) quite short. Tarsus (ta) of midbody legs quite short; as long as length of femur+postfemur; first two leg-pairs without an apical spine; leg-pair 1 with 1 ventral spine; leg-pair 2 with 3 ventral spines; leg-pair 3 with 4–6 ventral spines and 1 apical spine; leg pairs 4 with 6–8 ventral spines and 1 apical spine; leg pairs 5–21 with 7–9 ventral spines and 1 apical spine. In leg 9, length of femur equal to width, tarsus 4× longer than wide. Claw normal, at base with a notch, conspicuous.

Male sexual characters (Fig. 11F): Gonopore large, with long setae; with divided sclerotized plates, triangular.

Anterior telopods (Fig. 12A, B, E–G): First telopoditomere stout, rectangular. Telopoditomere 2 large. Immovable finger (process of telopoditomere 2) quite long; as long as telopoditomere 3; clearly seen in posterior and anterior views; strongly curved; tip obtuse, directed anteriad and close to basal part of telopoditomere 4; with a large membranous lobe; with sclerotized spots located at inner margin. Telopoditomere 3 longer than telopoditomere 4; clearly demarcated from telopoditomere 4 by conspicuous suture; apically with crenulated teeth (cr-t). Tepoloditomere 4 apically with a few setae located in the side pits.

Figure 12. 

Zephronia macula sp. nov., ♂ paratype (CUMZ-MYR0016) — SEM A–D overview of telopods (anterior view, lateral view with emphasis on anterior telopod, posterior view and ventral view, respectively) E–G anterior telopod (telopoditomeres 2–4, movable finger, and crenulated teeth, respectively) H–J posterior telopod (sclerotize spots on immovable finger, telopoditomeres 2–4 with emphasis on membranous lobe and crenulated teeth, respectively). Abbreviations: cr-t = crenulated teeth, ml = membranous lobe, scl-s = sclerotized spots. Scale bars in millimetres.

Posterior telopods (Figs 12A–D, H–J; 13): With 4 telopoditomeres. First telopoditomere rectangular, stout; slightly longer than wide. Telopoditomere 2 large and stout. Immovable finger (process of telopoditomere 2) long and slender, 3× longer than wide; equal in length to movable finger (= combination of telopoditomeres 3+4); attenuate near tip; tip obtuse, in-situ curving anteriad; inner margin with conspicuous sclerotized spots (scl-s) in a blackish ledge and two large membranous lobes (ml). Telopoditomere 3 long, 2.5× longer than wide, inner margin with a long membranous ledge and basally with a single spine. Telopoditomere 4 quite short; 3.5× shorter than telopoditomere 3; slightly tapering toward apex; with two spines located on membranous ledge at inner margin; with 9 or 10 large crenulated teeth (cr-t) posteriorly. Telopoditomeres 1 mostly glabrous, telopoditomere 2 in posterior view mostly glabrous, in anterior view covered by setae. Telopoditomeres 3 and 4 in posterior view glabrous; at basal part of telopoditomere 3 in anterior view sparsely setose. Inner horns with sharp-edged tips, slightly curved caudad.

Figure 13. 

Zephronia chantaburiensis sp. nov., ♂ paratype (ZFMK-MYR13660), drawings — left posterior telopod A anterior view B posterior view. Abbreviations: cr-t = crenulated teeth, imf = immovable finger, ml = membranous lobe. Scale bar: 0.5 millimetres.

Figure 14. 

Endoterga of thoracic shields and midbody tergites — SEM A–D Zephronia chantaburiensis sp. nov. (♂ paratype, CUMZ-MYR0014) E–H Zephronia macula sp. nov. (♂ paratype, CUMZ-MYR0016) A, B, E, F thoracic shields C, D, G, H midbody tergites. Abbreviations: cp = cuticular impression, ia = inner area, ma = middle area, oa = outer area, pm = posterior margin. Scale bars in millimetres.

Figure 15. 

Distribution and localities of Zephronia species in Thailand. Number inside the circle indicates species: 1 = Z. chantaburiensis sp. nov.; 2 = Z. chrysomallos; 3 = Z. enghoffi; 4 = Z. erawani; 5 = Z. golovatchi; 6 = Z. lannaensis; 7 = Z. macula sp. nov.; 8 = Z. panhai; 9 = Z. phrain; 10 = Z. siamensis; 11 = Z. viridisoma.

Female sexual characters (Fig. 11J, K): Vulva large and stout, sparsely setose; covering almost 1/3 of coxa; located at mesal margin; mesally extended to base of prefemur. Operculum (op) broad and well-rounded; swollen dorsoventrally, mesal margin not protruding; tip of operculum truncate. Bursa (bu) large, completely demarcated from operculum by a triangular groove.

Remarks

Intrapopulational variation was found in which most specimens have a brown/reddish brown (majority) colour, while the others are greenish brown (minority). In addition, the posterior margin of subanal scale is in most specimens wide, whereas some specimens possess a quite narrow margin.

Distribution and habitat

This species is widely distributed across eastern Thailand (>150 km). Most specimens were collected from locations with granitic rock habitats, while a few localities were in forested limestone habitats (Fig. 8H, I). All individuals were encountered during the day under rotten logs and in leaf litters.

Etymology

The name is a noun referring to the pattern of spots on the body.

Discussion

Two new species of giant pill-millipedes from Thailand have been integratively described on the basis of morphological characters and genetic information. These new species are taxonomically assigned to the genus Zephronia due to the combination of the distinct characters of the antennae, endotergum, tarsi of legs 5–21, anterior telopods, posterior telopods and female vulvae; they share a few morphological traits with most of their congeners (viz., body size ca 20 mm with brown colour, one row of bristles on endotergum). They clearly belong to the Zephronia s.s. species group (Z. chrysomallos, Z. dawydoffi, Z. erawani, Z. enghoffi, Z. golovatchi, Z. hui, Z. konkakinhensis, Z. lannaensis, Z. laotica, Z. medongensis, Z. montis, Z. ovalis, Z. panhai, Z. siamensis and Z. zhouae), both morphologically and genetically (Fig. 1). However, Z. chantaburiensis sp. nov. and Z. macula sp. nov. obviously differ from other Zephronia s.s. species in the presence of only a single apical spine on the tarsi, as well as in the anterior telopods (having a relatively long and strongly curved immovable finger). Among the Zephronia species in Thailand, the presence of a single apical spine on the tarsi is only known for Z. viridisoma, a species genetically distant from most Zephronia species (Fig. 1). One of the new species, Z. macula sp. nov., exhibits a distinct colour pattern by having spots/bands throughout the body tergites, which is unique for the genus, but similar to the one observed in Sphaerobelum nigrum Wesener, 2019 (Wesener 2019).

The interspecific distances based on the 658 bp COI barcoding fragment of the two new species compared to its congeners are quite large, with 18.94–26.82% in Z. chantaburiensis sp. nov. and 17.93–25.13% in Z. macula sp. nov. While the usual range of interspecific genetic distances for species discrimination in most giant pill-millipedes is from 8 to 21%, the observed distances in this study are higher than between most previously recognized species within the genus. Our analysis of the interspecific distances is consistent with those reported in recently described Zephronia species (Rosenmejer et al. 2021; Bhansali and Wesener 2022), which have interspecific distances ranging from 8–15%. The relatively high maximum interspecific distances observed between millipede species have been proposed to result from isolated distributions with different degrees of geographical barriers, because often closely related species are found geographically far from one another (Cádiz et al. 2018; Means and Marek 2017). The geographical distances between the new species described here and other Zephronia species also support the discrimination of our new species. Although our single-locus phylogenetic tree placed our two new species into the Zephronia s.s. (Fig. 1), the deep evolutionary relationships and the monophyly of this widespread and diverse genus, especially Zephronia s.s. species group, are still unclear. An integrative approach implementing additional genetic markers such as nuclear genes (e.g. see Dietz et al. 2023), together with the inclusion of more taxa should be pursued to clarify their generic-species status.

We conducted four intensive surveys throughout the eastern part of Thailand from 2019 to 2023. According to the distribution record, Z. macula sp. nov. has a wide range, covering the majority of eastern Thailand (Fig. 13). The wide distribution is similar to those of Z. siamensis and Z. phrain, which are dispersed over an area stretching 200–300 km2 (Srisonchai et al. 2021). The wide distribution of Z. macula sp. nov. is probably due to its occurrence in a wide range of granite and limestone habitats, where it is easily encountered, whereas Z. chantaburiensis sp. now. has been collected only in areas with granitic soils. It is important to note that Z. chantaburiensis sp. nov. lives in sympatry with Z. macula sp. nov. by sharing the same habitat/ microhabitat; individuals of both species can be found under leaf litter and rotten logs. Quite often, several millipede species of the same genus can be found in the same location, e.g., in Zoosphaerium Pocock, 1895; Eviulisoma Silvestri, 1910; Chaleponcus Attems, 1914 (Wesener and Sagorny 2021; Enghoff 2014, 2018). Given that the two new species were only found in the eastern part of Thailand, we here regard them as endemic.

The present work adds two endemic species of Zephronia, resulting in a total of 11 species for the genus in Thailand (51 valid species worldwide). This discovery has also greatly expanded the known range of the genus in the far eastern part of Thailand, but leaves a gap of more than 400 km without any record of Zephronia along the coast of Thailand in the closest country area (Cambodia) where the Cardamom Mountain Range lies. Searching in still unexplored places in Thailand and neighboring counties would be fruitful in revealing the hidden diversity of the genus and the evolution of the taxon. It is believed that as investigations and intensive analyses go further, more new species will certainly be discovered.

Acknowledgements

The curators and staffs of NHMW (Nesrine Akkari), ZFMK and NHMD (Henrik Enghoff) are acknowledged for allowing access for type examinations. We thank the students of Khon Kaen University Museum of Zoology (KKUMZ) and the members of Animal Systematic Research Unit (ASRU), Chulalongkorn University for all their assistance in collecting materials. Many thanks also go to Khanet Srikampha for SEM preparations and Dave Anderson for English corrections. We are grateful to the editor Nesrine Akkari and the referees Henrik Enghoff, Dragan Antic, Sergei Golovatch, and Nikolaus Szucsich for their critical comments which improved the manuscript. The CIPRES Science Gateway V 3.3 provided access to computational analysis.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work (Grant No. RGNS65-056) was supported by the Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (OPS MHESI), Thailand Science Research and Innovation (TSRI), and partly granted by the National Science, Research and Innovation Fund of Khon Kaen University. RS’s visit to the NHMD, NHMW and ZMFK was supported by the Academic Affairs Promotion Fund, Faculty of Science, Khon Kaen University, Fiscal year 2024 (RAAPF).

Author contributions

Conceptualization: TW, RS. Data curation: RS, TW. Formal analysis: RS. Funding acquisition: RS. Investigation: RS. Methodology: TW, RS. Project administration: RS. Resources: NL, CS, RS. Supervision: CS, TW. Validation: CS, TW. Visualization: RS, NL. Writing - original draft: RS. Writing - review and editing: RS, TW, CS.

Author ORCIDs

Ruttapon Srisonchai https://orcid.org/0000-0002-7142-0999

Natdanai Likhitrakarn https://orcid.org/0000-0002-1306-317X

Chirasak Sutcharit https://orcid.org/0000-0001-7670-9540

Thomas Wesener https://orcid.org/0000-0002-2028-3541

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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Supplementary materials

Supplementary material 1 

Estimates of cytochrome c oxidase I (COI) sequence divergences (uncorrected p-distances) within and among Zephronia species and related genera (Sphaerobelum, Prionobelum and Cryxus)

Ruttapon Srisonchai, Natdanai Likhitrakarn, Chirasak Sutcharit, Thomas Wesener

Data type: xlsx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (86.41 kb)
Supplementary material 2 

Phylogenetic tree based on maximum likelihood (ML) approach of genus Zephronia and related genera (Sphaerobelum, Prionobelum and Cryxus)

Ruttapon Srisonchai, Natdanai Likhitrakarn, Chirasak Sutcharit, Thomas Wesener

Data type: pdf

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (9.58 kb)
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